DESCRIPTION: (Applicant's Abstract) Cysteine-string proteins (csps) are a novel family of synaptic vesicle proteins that have been implicated in two important processes at nerve endings. First, recent evidence strongly supports the hypothesis that csps are part of a unique regulatory interaction that takes place between a docked synaptic vesicle and presynaptic calcium (Ca) channels. The functional consequence of this interaction is that the presynaptic Ca channels are rendered competent to open in response to membrane depolarization. However, the molecular and biophysical mechanisms of this interaction remain unknown. Because csps associate with a specific isoform of Hsp70 molecular chaperones, we have hypothesized that the csp-Ca channel link involves a specific isoform of Hsp70. Specific Aim 1 directly tests this hypothesis using both biochemical and physiological approaches. Second, we have postulated that csps participate in events that are necessary for the fusion of synaptic vesicles with the plasma membrane. The rationale for this proposal stems from a consideration of the unusual structure of csps. The cysteine string domain of these proteins contains as many as 11 consecutive cysteine residues. As far as we know, all of these cysteine residues are fatty acylated. A protein with this type of a hydrophobic domain can assume a conformation at membrane interfaces that can effectively cross-link adjacent membranes (a model of this interaction is presented). Specific Aim 2 tests the validity of this model of membrane cross-linking by studying the effect of csps on the aggregation, fusion or lysis of liposomes under different empirical conditions. Finally, Specific Aim 3 considers several inter-related structural studies of csp that are relevant to its presumed functions. Among the planned studies are efforts: (I) to resolve unequivocally the degree of fatty acylation of native csps; (ii) to examine the secondary structure of membrane-associated csps; and (iii) to use a protein palmitoylthioesterase to probe the role of membrane tethering and palmitoyl residues in csp function at nerve endings. In summary, these investigations will advance our knowledge of csps on several fronts that are germane to its function at synapses. Because csps are widely distributed at nerve endings and in other secretory cells, this work is of fundamental importance to our understanding of membrane trafficking events in normal and pathological circumstances.